DEVELOPMENT OF EXPRESSION CLONING SYSTEM FOR ONCOGENE CDNAS

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We are developing a cDNA expression cloning system for isolation of dominant as well as recessive oncogenes. ln this system. cDNA libraries are constructed in eukaryotic expression vectors using poly(A)-selected RNAs from transformants or tumors. The library DNA can then be used to transfect NIH/3T3 cells. cDNA clones will be recovered from resulting foci, and their structures analyzed. In order to make expression cloning feasible for this purpose, cDNA libraries containing complete coding sequences will be necessary. At this point, we have developed a high efficiency cDNA cloning system which can direct the orientation of inserts in eukaryotic expression vectors which possess large cloning capacities. Our lambda-plasmid composite vectors contain a retroviral long terminal repeat (LTR) promoter to express cDNA and a simian virus 40 (SV40) early promoter-driven neo gene as a eukaryotic cell selection marker. cDNA was synthesized from a linker-primer containing the site for SfiI, an infrequent cutter of DNA. An adaptor was ligated at both ends of the double-stranded cDNA molecules, cleaved by SfiI, and ligated with the lambda vector arms prepared by cutting at two different SfiI sites. Due to the directional cloning strategies and non-symmetrical structure of sticky ends of both the vector and insert DNAs, the efficiencies of 10 to 100 million plaque-forming units of phages were obtained from one microgram of poly(A)-selected RNA. Furthermore, we have shown that our libraries contain cDNAs for several growth factors and receptors that are nearly full length molecules of 2.5 to 6.5 kb, at high frequencies.